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Incorporating 6 NNK codons corresponds to about 1 billion possible combinations, and 18 N mixed bases will create a pool with 68.7 billion different gene fragments. As the number of variable bases increases, the number of molecules representing a particular sequence decreases. We decided to limit variable regions to 18 mixed bases to give customers the best overall pool of library constructs. Since most functional screens cover 1,000–100,000 recombinant colonies, allowing 18 mixed bases should be adequate for producing meaningful results.